Screening and validation of double allele-specific binding F-primers for the measurement of antihypertensive pharmacogenomics

Objective Previous studies have proposed that genetic polymorphisms of CYP2D6*10, ADRB1, NPPA, CYP3A5*3, ACE, CYP2C9*3, and AGTR1 are involved in antihypertensive pharmacogenomics. The purpose of this study is to develop an amplification analysis using double allele-specific (AS) binding primers for accurate measurement of antihypertensive pharmacogenomics. Methods To establish a quadruplex quantitative PCR (qPCR) analysis for genotyping of CYP2D6*10, ADRB1 (1165 G>C), NPPA (2238 T>C) and CYP3A5*3, and a triplex qPCR analysis for genotyping of ACE (I/D), CYP2C9*3 and AGTR1 (1166 A>C), mismatch AS F-primers were screened by detection of plasmid/gDNA, and were validated by agreement analysis/reproducibility evaluation, in which the ΔCq (differences in threshold cycles between the wild-type F-primer-based amplification assay and the mutant-type F-primer-based amplification assay) was employed to determine genotypes. Results Seven pairs of primers were successfully selected through three rounds of F-primers screening. Except for ADRB1, the robustness assessment showed the amplification efficiency ranging from 0.9 to 1.1. In agreement analysis, two specimens in the training set (n = 203) were defined by the triplex analysis rather than NGS as heterozygotes for ACE, which was evidenced by gel electrophoresis. Reproducibility evaluation demonstrated that the coefficient of variation (CV) was <5%. Conclusion Multiplex amplification analysis using screened AS binding primers is a simple, reliable, and accurate tool to guide drug delivery in antihypertensive personalized treatment.


Introduction
Hypertension is becoming the main cause of cardiovascular disease (1)(2)(3)(4).Patients with hypertension reached 1.278 billion worldwide in 2019 (5).A previous study reported that intensive hypertension control could avoid 2.209 million coronary heart disease (CHD) events, 4.409 million stroke events, and 75,100 cardiovascular deaths in 10 years (6).However, hypertension control is still poor because of insufficient clinical experience and apparent drug resistance (2).

FIGURE
Design strategy of this study.F-primers as polymorphism-binding oligonucleotides were optimized through three rounds of screening and validated with agreement analysis considering next-generation sequencing as a reference method.
First, F-primers were screened by plasmid determination using uniplex qPCR.Second, selected F-primers were screened by detecting genomic DNA using multiplex qPCR, including a quadruplex analysis for CYP2D6 * 10, ADRB1, NPPA, and CYP3A5 * 3 and a triplex analysis for ACE (I/D), CYP2C9 * 3, and AGTR1.Each of the multiplex analyses contained two reactions: the wild-type F-primer-based amplification assay and the mutanttype F-primer-based amplification assay, in which Cq (differences in threshold cycles between the wild-type F-primer-based assay and the mutant-type F-primer-based assay) was employed to determine genotypes.Third, the F-primers were screened by robustness assessment.Finally, the screened F-primer-based assay was validated by concordance analysis, using NGS as the reference method.The sterilized distilled water instead of DNA was used as the negative control during the whole experiment process.The design strategy of this study is shown in Figure 1.

. Sample collection and DNA extraction
A total of 203 oral swab samples were collected from Chinese volunteers.Genomic DNA was isolated using the QIAamp DNA Mini kit (Cat No.51304, QIAGEN, Dusseldorf, Germany), according to the manufacturer's instructions.The quality and quantity of DNA were determined by the NanoPhotometer P360 (Implen GmbH, Munich, Germany).Sanger sequencing was conducted by Personal company (Qingdao Personal Biotechnology Co., Ltd., China).NGS sequencing was conducted by the Center for Molecular Diagnosis at Shandong Provincial Hospital, affiliated with Shandong First Medical University (Jinan, China).

. Primers and probe design
Based on the nucleotide sequences of these seven genes, AS F-primers, co-reverse primers, and hydrolysis probes were designed using Primer Express 3.0.The probes for CYP2D6 * 10, ADRB1 (1165 G>C), NPPA (2238 T>C), and CYP3A5 * 3 in the quadruplex analysis were labeled with the fluorescent dyes FAM, VIC, NED, and CY5 at their 5 ′ ends and the quencher BHQ1, BHQ1, BHQ2, and BHQ3 at their 3 ′ ends, respectively.The probes for ACE (I/D), CYP2C9 * 3, and AGTR1 (1166 A>C) in .
The uniplex amplification analysis was conducted according to the same protocol.The qPCR protocols started with a contamination digestion step for 2 min at 37 • C, and a pre-denaturation step for 5 min at 95  .

Data analysis
Data analysis and graphing were carried out using GraphPad Prism software version 9 (GraphPad Software, Inc., San Diego, CA).

Results
. First round of F-primers screening by measurement of plasmid using uniplex qPCR AS F-primers with the second, third, or fifth 3 ′ -terminal mismatched base, co-hydrolysis probes, and reverse primers were designed for each SNP.Uniplex qPCR analysis and plasmid  models covering homozygotes and heterozygotes were employed to screen mismatch AS forward primers (n = 72) to roughly enable maximization of Cq (differences in threshold cycles between the wild-type F-primer-based assay and the mutated-type F-primerbased assay) (Supplementary material S1).The originally selected F-primers are shown in Table 1.
. Second round of F-primers screening by detection of gDNA using multiplex qPCR Selected F-primers were in succession screened by examination of human gDNA, comprising homozygotes and heterozygotes.To omit the positive control set in antihypertensive pharmacogenomic measurement, positive outcomes obtained from the wild-or mutated-type F-primer-based assay were required.Because undetermined results were observed in gDNA scans, the concentration of mutated-type F-primer for AGTR1 was adjusted from 0.2 to 0.4 µM to ensure positive outcomes (Figure 2).

. Third round of F-primers screening by robustness assessment
Six concentrations (40 ng, 20 ng, 10 ng, 5 ng, 2.5 ng, and 1.25 ng) of heterozygotes from oral swabs were prepared.Amplification efficiency was calculated using the generated calibrator curve: 10 −1/slope −1, with the logarithm of the template copies plotted on the X-axis and Cq plotted on the Y-axis (24).The reactions were conducted in duplicate with three dependent experiments.As the calibrator curve did not appear in a dose-dependent manner, Fprimer WF2/MF2, probe P2, and reverse primer R2 for ADRB1 were substituted for F-primer WF1/MF1, probe P1, and reverse primer R1, respectively, in the quadruplex amplification analysis (Table 1 and Figure 3).Except for ADRB1, optimized calibrator curves demonstrated amplification efficiencies ranging from 0.9 to 1.1 and analytical sensitivities of at least 1.25 ng (Figure 4). .

Verification of screened F-primers-based analysis by agreement analysis
Considering NGS as a reference method, we examined 203 gDNA samples extracted from oral swabs to evaluate the accuracy of the multiplex analyses using double allele-specific binding Fprimers.The results showed that, besides ACE, the coincidence rate was 100%.Two specimens (No.001 and No.056) in the training set (n = 203) were defined by the analysis rather than NGS as heterozygotes for ACE, which was evidenced by gel electrophoresis (Figure 5).The cutoff values for genotyping are shown in Table 2.

. Substantiation of screened F-primers-based analysis by producibility evaluation
To evaluate the producibility of the analysis, each heterozygote was tested in eight-plicates by two operators, using two different reagent lots every 5 days (n = 80/specimen) at one site.A total of 80 Cq values were collected to calculate the coefficient of variation (CV).The results showed that the CV values for reproducibility were within 4.00% for all days, specimens, replicates, operators, and reagent lots combined (Figure 6).

Discussion
In this study, multiplex amplification analysis was established for the measurement of hypertensive pharmacogenomics.Due to genetic polymorphism, only about one-third of patients with hypertension accept effective treatment (25-30).Therefore, this study is helpful for hypertension patients to take more effective and well-tolerated medication.
Compared to other methods (31), multiplex qPCR behaves as a simple and effective approach to the detection of pharmacogenomic SNPs (32).Polymorphism-specific binding molecules in PCR-based analysis comprise dsDNA-binding dye, AS probe, and primer (33, 34).The dsDNA-binding dye-based high-resolution dissolution curve (HRM) assay needs a specific equipment module.In addition to the diseconomy, it is time-consuming and laborious to discover an appropriate minor groove binder (MGB) probe (35).The wildtype allele reaction probably outcompetes the mutated-type allele reaction when two AS probes barcoded with different fluorophores are utilized to identify genetic polymorphism (36).Sometimes, it is difficult to accurately discriminate SNPs using single-color melting curve analysis.For enhancement of AS primer specificity, base mismatch is more economical than locked nucleic acid (LNA) decoration (37).In this study, mismatch AS primers were screened and validated.The combination of a wild-type AS F-primerbased amplification assay with a mutated-type AS F-primer-based amplification assay was utilized to obtain Cq to define the genotype.The results evidenced that screened F-primer-based amplification analysis is a simple, accurate, and reliable approach to measure antihypertensive pharmacogenomics.
As the definition of ACE (I/D) genotype for two specimens differed between triplex analysis and NGS, we utilized PCRgel electrophoresis to substantiate the outcomes.Located on chromosome 17, the ACE gene consists of 26 exons and appears as a polymorphism in the form of either insertion (I) or deletion (D) of a 287-bp Alu repeat sequence in intron 16.The ACE (I/D) allele can be detected by PCR using the primers flanking the 287 bp insertion sequence (38).In gel electrophoresis, the I/I genotype can be identified by the presence of a single 490 bp amplicon, the D/D genotype can be recognized by the presence of a single 190 bp product, and the I/D genotype extends both 490 and 190 bp amplicons (39).The results of gel electrophoresis validated the accuracy of the triplex analysis for ACE (I/D) measurement (Figure 5).
Based on hypertensive pharmacogenomics of CYP2C9 * 3, ADRB1(1165 G>C), AGTR1 (1166 A>C), CYP2D6 * 10, ACE (I/D), CYP3A5 * 3, and NPPA (2238 T>C), the principle of personalized drug delivery was proposed as follows: (a) doubling the standard dose is suggested when the hypertension is   moderately sensitive to certain anti-hypertensive drugs; and (b) the minimum dose is recommended to initiate treatment when the hypertension is highly sensitive to certain antihypertensive drugs (7).Following the above principle, clinical studies evidenced that, compared to clinic experience-guided anti-hypertensive therapy, genotype-guided treatment appeared more effective and had fewer side effects.Herein, we established a simple, efficient, and accurate method for simultaneously detecting the genotypes of CYP2D6 * 10, ADRB1 (1165 G>C), NPPA (2238 T>C), CYP3A5 * 3, ACE, CYP2C9 * 3, and AGTR1 (1166 A>C) by screening and verification of mismatched AS Fprimers.

Conclusion
As an accurate and reliable approach, the analysis described in this study is a valuable tool to determine the genotypes for CYP2D6 * 10, ADRB1, NPPA, CYP3A5 * 3, ACE, CYP2C9 * 3, and AGTR1, which can guide drug delivery in antihypertensive treatment to ensure curative effect.Employing the similar technique verified in this study, our laboratory will design and develop a multiplex amplification analysis for guiding aspirin delivery in the future.

FIGURE
FIGUREAmplification plots of AGTR in the triplex amplification analysis.The concentration of mutated-type F-primer for AGTR was adjusted from .(A) to .µM (B) to ensure positive outcomes.Wild-type homozygote was detected in duplicate by the triplex amplification analysis.

FIGURE
FIGUREAmplification plots of ADRB in robustness assessment.To improve the robustness of ADRB measurement, F-primer WF /MF , probe P , and reverse primer R were substituted for F-primer WF /MF , probe P , and reverse primer R , respectively, in the quadruplex amplification analysis.(A) Unoptimized amplification plots.(B) Optimized amplification plots.To assess robustness, serial dilutions of heterozygote ( .-ng) were measured by the quadruplex amplification analysis, including the wild-type F-primer-based amplification assay and the mutated-type F-primer-based amplification assay.Reactions were run in duplicate.

FIGURE
FIGURE Robustness of optimized amplification analysis.The robustness assessment was executed by employing mismatch allele-specific F-primers targeting single nucleotide polymorphisms to simultaneously detect heterozygotes.Serial dilutions of heterozygotes ( .-ng) were measured by multiplex amplification analysis containing a wild-type F-primer-based amplification assay and a mutated-type F-primer-based amplification assay.(A) (a) Calibrator curves of quadruplex amplification analysis.Amplification e ciency (E ) % and R are shown; (b) Amplification plots of the robustness assessment in quadruplex amplification analysis.Representative amplification plots are shown.(B) (a) Calibrator curves of triplex amplification analysis.Amplification e ciency (E ) % and R are shown; (b) Amplification plots of the robustness assessment in triplex amplification analysis.Representative amplification plots are shown.Reactions were run in duplicate with three independent experiments.Data are expressed as mean ± SE.DF, deletion-type F-primer; IF, insertion-type F-primer; WF primer, wild-type F-primer; MF primer, mutated-type F-primer; Het, heterozygote.

FIGURE
FIGURE Gel electrophoresis of PCR amplicon for ACE.The D/D genotype was identified by the presence of a single -bp amplicon, and the I/D genotype extended both -bp and -bp amplicons.The ACE genotypes of controls and were D/D and I/D, respectively, defined by Sanger sequencing.M, DNA marker; I/D, heterozygote; D/D, deletion homozygote.

FIGURE
FIGURE Producibility evaluation.(A) The inter-day CV values for the wild-type F-primers-based amplification assay in quadruplex analysis.(B) The inter-day CV values for the mutated-type F-primers-based amplification assay in quadruplex analysis.(C) The inter-day CV values for the wild-type F-primers-based amplification assay in triplex analysis.(D) The inter-day CV values for the mutated-type F-primers-based amplification assay in triplex analysis.The inter-day CV value was < % for all days, specimens, replicates, operators, and reagent lots combined.
TABLE Primer and probe sequences.35 s.The fluorescence signal was collected at 60 • C.These amplifications were performed on the ABI7500 Real-Time PCR Instrument (Thermo Fisher Scientific Inc., MA, USA).